A number of studies have conventionally been known on the fixation of physiologically active substances on carriers in the solid phase. The main stream of these studies today exists in the fixation of enzymes, although there are many experiments of such kind, including: fixation of immunoreactive substances such as antigens and antibodies for the purpose of obtaining materials for medical treatment; adsorption of particular physioactive substances by means of separation materials in the affinity chromatography; and fixation of tangible components such as useful fungous bodies and cells for use in bioreactors and/or artificial organs.
The word "fixation" herein used indicates that these physioactive substances, originally water-soluble, are made water-insoluble without at all affecting their physiological functions. There are various approaches to the fixation, including carrier bonding, bridging, and integration methods. Of these, the carrier bonding method, which achieves the fixation of a substance through a covalent bond with a carrier, provides the stablest result with the highest bonding power and accordingly with the least disengagement. One of many reported examples of this method is diazo-bonding of a protein (e.g. albumin) for the fixation thereof on a diazonium compound produced by adding dilute sulfuric acid and sodium nitrate to a water-insoluble carrier (R--NH.sub.2) containing an amino group. (Chambell et al., Proc Nat Acad Sci , 37, 575 (1951))
Meanwhile, as an improvement of this carrier bonding method, a straight chain structure called a "spacer" or "arm" containing an n-alkyl chain has recently been introduced between the carrier and the physiologically active substance to be bonded. This is because the introduction of such structure allows the physiologically active substance to be fixed at an end of the structure, which reduces steric infection generated form the reaction of the substance with the substrate, thus facilitating it to manifest its function.
Kim et al., for example, fixed heparin, an anti coagulant, on agarose beads containing an n-alkyl chain (wherein n is 2, 4, 8, 10, or 12) as the spacer, and found that the anticoagulating activity measured by the activated part thromboplastin time (APTT) becomes larger with a longer alkyl chain. This phenomenon was named the "spacer effect".
(Kim et al., Thrombosis Research, 26, 43 (1982))
However, a hydrophobic spacer such as an n-alkyl chain has a lower affinity for the physiologically active substances which are essentially water-soluble, and accordingly, the fixation rate may be reduced, or denaturation or deactivation of the physiologically active substances may be caused by the fixation. In addition, since a long n-alkyl chain is water-insoluble without respect to the end functional group, it is necessary to use an organic solvent in order to introduce it into the carrier, and the organic solvent to be used should be selected from an extremely limited range because some carriers are dissolved, denaturated, or damaged by some organic solvents.
Furthermore, on the surface of the carrier which is hydrophobicized by the introduction of such hydrophobic spacer, non-specific adsorption and/or denaturation of other physiologically-active substances, in particular of proteins, tend(s) to occur. This may affect the essential function of the physiologically active substances fixed.
A purpose of this invention is to provide fixing materials for use with physiologically active substances capable of highly revealing physioactive functions thereof when fixed. Another purpose of this invention is to provide fixed physiologically active substances which are fixed by said fixing materials and which have high activity.